Oncotarget, Vol. 7, No. 25
www.impactjournals.com/oncotarget/
Research Paper
MARCKS contributes to stromal cancer-associated fibroblast activation and facilitates ovarian cancer metastasis Zongyuan Yang1,*, Sen Xu1,*, Ping Jin1, Xin Yang1, Xiaoting Li1, Dongyi Wan1, Taoran Zhang1, Sixiang Long1, Xiao Wei1, Gang Chen1, Li Meng1, Dan Liu1, Yong Fang1, Pingbo Chen1, Ding Ma1, Qinglei Gao1 1
ancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, C Wuhan, Hubei 430030, China
*
These authors have contributed equally to this work
Correspondence to: Ding Ma, email: [email protected] Qinglei Gao, email: [email protected] Keywords: ovarian cancer, CAFs, MARCKS, senescence, Twist1 Received: January 07, 2016 Accepted: March 28, 2016 Published: April 13, 2016
ABSTRACT The Cancer Genome Atlas network has revealed that the ‘mesenchymal’ epithelial ovarian cancer (EOC) subtype represents the poorest outcome, indicating a crucial role of stromal cancer-associated fibroblasts (CAFs) in disease progression. The cooperative role of CAFs in EOC metastasis has long been recognized, but the mechanisms of stromal CAFs activation are still obscure. Therefore, we carried out an integrative analysis to identify the regulator genes that are responsible for CAFs activation in microdissected tumor stroma profiles. Here, we determined that myristoylated alanine-rich C-kinase substrate (MARCKS) was highly expressed in ovarian stroma, and was required for the differentiation and tumor promoting function of CAFs. Suppression of MARCKS resulted in the loss of CAF features, and diminished role of CAFs in supporting tumor cell growth in 3D organotypic cultures and in murine xenograft model. Mechanistically, we found that MARCKS maintained CAF activation through suppression of cellular senescence and activation of the AKT/ Twist1 signaling. Moreover, high MARCKS expression was associated with poor patient survival in EOC. Collectively, our findings identify the potential of MARCKS inhibition as a novel stroma-oriented therapy in EOC.
“proliferative,” and “mesenchymal,” in a cohort of 557 serous EOC patients [5]. A subsequent follow-up study determined that patients with the “mesenchymal” subtype presented the worst prognosis [7]. These newly emerged classification schemes based on molecular profiling facilitate our understanding of EOC heterogeneity and the development of personalized treatment strategies [8-10]. Moreover, this advantageous stratification emphasized the importance of tumor microenvironment, especially in terms of the stromal infiltrating components in EOC patients. The most prominent cell types in the tumor microenvironment are the cancer-associated fibroblasts (CAFs), which primarily contributed to the assignment of the “mesenchymal” cluster [11, 12]. CAFs are heterogeneous populations that include myofibroblasts and reprogrammed variant normal tissue derived cells such as
INTRODUCTION High-grade epithelial ovarian cancer (EOC) remains the most lethal gynecological cancer and exhibits considerable heterogeneity [1]. Traditional histopathological classification of EOC into “serous,” “mucinous,” “clear cell,” and “endometrioid” subtypes was limited in guiding therapy decisions [2, 3]. To complement conventional histopathology, molecular classification based on large-scale gene profiling was carried out and enabled the discovery of several EOC subtypes [4-6]. Tothill et al. first reported six molecular categories in 285 EOC patients and found that tumors expressing a reactive stromal gene signature were associated with a poor prognosis [4]. More recently, The Cancer Genome Atlas (TCGA) project described four subtypes, namely, “immunoreactive,” “differentiated,” www.impactjournals.com/oncotarget
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fibroblasts and endothelial and mesothelial cells in EOC [13-15]. Generally, CAFs support cancer cells through both cell-to-cell contact interactions and soluble factors via secretion of cytokines, chemokines and ECM (Extracellular matrix) components [16-18]. The cooperative role of CAFs in EOC cell proliferation, adhesion and metastasis has long been recognized, but the mechanisms involved in stromal CAF activation are still largely unknown [18-20]. Previous studies have emphasized that perpetual activation of stromal CAFs is indispensable for tumor expansion [14-19]. Therefore, understanding the molecular profile of activated CAFs could help in targeting this major accessory of tumor microenvironment. Recent molecular investigation has identified a “stromalresponse” signature that predicts poor prognosis [21] and defined a “reactive stroma signature” characterizing primary chemoresistance in EOC [22]. Although previous studies sought to characterize specific ovarian tumor stromal genes in a compartmentalized fashion, the samples used were whole tumor specimens, which cannot exclude the interference of the epithelial compartment. Meanwhile, the recently discovered stromal signature genes are more likely to be downstream functional genes than the upstream regulator genes. These factors prompted us to explore the underlying regulators that control the active CAF signature in pure tumor stromal tissues. The emergence of specialized microdissected stroma profiling data allows the identification of tumor stroma gene signatures, as well as the potent regulator genes controlling stroma activation [18, 23-25]. To identify the regulators of stromal CAF activation, we employed the most widely accepted marker of the CAF phenotype— αSMA, which constitutes a stress fiber system bridging communication between CAFs and the ECM [26]. Thus, we carried out an integrative analysis of microdissected stromal gene profiles of EOC and invasive breast tumors [18, 25]. Among the genes identified, myristoylated alanine-rich C-kinase substrate (MARCKS) was found to be highly associated with αSMA expression in EOC tumor stroma, and was notably overexpressed in tumor stroma of both ovarian and invasive breast cancer. MARCKS, originally identified as a major target of protein kinase C (PKC), is a key regulatory molecule regulating actin dynamics [27]. Recently, it has been shown to play a fundamental role in mediating chemoresistance of breast and lung cancer [28, 29]. There are limited studies examining MARCKS in EOC metastasis. Although MARCKS has been reported to promote fibroblast migration [30], the role of MARCKS on CAF traits and the underlying mechanism involved is not well understood. This study demonstrates elevated stromal expression of MARCKS along with OC advancement, and shows that MARCKS sustains the CAF features through suppression of cellular senescence and maintenance of AKT signaling. Suppression of MARCKS attenuates CAF activity and www.impactjournals.com/oncotarget
their tumor-supporting role in 3D organotypic culture and an OC murine xenograft model. A meta-analysis of a total of 2970 serous OC expression profiles confirmed MARCKS as a prognostic factor of poor patient outcome. Our results address the role of MARCKS in the tumor stroma as a pivotal regulator of CAF activation. Thus, MARCKS could be an attractive target for stroma-oriented therapy in EOC patients.
RESULTS Overexpression and significance of MARCKS in tumor stromal fibroblasts To explore the regulatory molecules that drive gene expression representative of CAF features in EOC, we carried out an integrative analysis to identify genes that are important for CAF activation and specifically upregulated in tumor stroma. Currently, the most definitive molecular marker of CAFs is αSMA, which indicates activation of normal fibroblasts and plays a critical role in mediating communication between the stromal cells and the matrix [26, 31]. Here, we identified a cluster of 503 genes (Supplementary Table S1) that were notably positively correlated with αSMA expression in dataset GSE40595 that includes microdissected ovarian profiling data, of which fibroblasts were shown to be the major constituent [32]. By analyzing overlapping genes with the calculated 784 genes (Supplementary Table S2) in ovarian tumor stroma and 468 genes (Supplementary Table S3) in breast tumor stroma that significantly upregulated compared with their normal fibroblast counterparts in GSE40595 and breast stromal profile GSE9014, we identified ARID4B, COL3A1 and MARCKS as candidate targets for controlling stromal activation (Figure 1A). Among the genes identified, ARID4B expression was not correlated with EOC patient survival (Supplementary Figure 1A and 1B). In contrast, higher COL3A1 expression was notably correlated with worse patient outcome (Supplementary Figure 1C and 1D). However, the role of collagen family members in remodeling ECM were well studied [33], and they were more likely to be the downstream functional executors than the upstream regulators of stromal activation. MARCKS was selected for further study due to its reported role in regulating tumor cell adhesion and migration [34-36]; its impact on CAFs activity has not been well studied. The advantageous expression of MARCKS in cancer stroma was further confirmed in another two stoma profiling datasets of lung and prostate cancer and in tumor stroma of EOC patient samples (Figure 1B and 1C). Compared with its expression in the tumor epithelial compartment, MARCKS was noted specifically expressed in the stromal compartment as determined by analysis of EOC-related datasets (Figure 1D). In contrast, MARCKS level was reduced in the tumor epithelial cells compared with normal ovary epithelial 37650
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tissues (Figure 1E). Data mining in EOC profiling data also showed that MARCKS expression was elevated along with disease metastasis (Figure 1F) or after chemointervention (Figure 1G). All these data demonstrated that MARCKS was highly expressed in ovarian stroma and might regulate the stromal CAF activity.
specimens [37]. We found that MARCKS expression was elevated in tumor stroma and reduced in tumor epithelia as OC progresses (Figure 2C), and was increased in patients with a higher FIGO stage (Figure 2D) or in those that developed chemoresistance or disease recurrence (Figure 2E). Next, we assessed the effect of tumoral MARCKS expression on patient survival. Firstly, we analyzed its significance in a large set of EOC samples included in curatedOvarianData [38], which includes expression data as well as survival information. MARCKS mRNA expression was found to be significantly correlated with poor patient overall survival (OS) (HR=1.11, p=1.35e-4) (Figure 2F), and with worse progression-free survival (PFS) in EOC patients (HR=1.07, p=0.0499) (Figure 2G). The impact of MARCKS on patients survival was further analyzed in a large annotated database of breast and lung cancer patient samples. Kaplan–Meier analyses showed that elevated MARCKS was significantly correlated with decreased OS (HR=1.28, p=0.039), post-progression survival (PPS) (HR=1.63, p=0.00066) and relapse-free survival (RFS) (HR=1.59, p=2.1e-14) (Supplementary Figure 2A) in breast cancer and with decreased OS (HR=1.3, p=5.7e-05), PPS (HR=1.46, p=0.008) and first progression (FP) (HR=1.95, p=1.5e-10) (Supplementary Figure 2B) in lung cancer. These findings indicated that
MARCKS is specifically expressed in stromal CAFs and correlates with patient outcome To further validate the expression pattern of MARCKS during OC progression, we analyzed MARCKS expression in tissue samples including 10 normal ovarian tissues, 12 normal fallopian tube tissues and 18 pairs of primary and metastatic EOC tissues. Immunohistochemistry (IHC) demonstrated that 65% of normal ovary and 72% of the fallopian tube tissue versus 18% of the primary and 27% of the metastatic tumor samples showed moderate to strong epithelial MARCKS immunostaining, whereas 68% of the primary and 95% of the metastatic tumor sample versus 14% of the normal ovary and 32% of the fallopian tube tissues showed moderate to strong stromal MARCKS immunostaining. (Figure 2A and 2B). Additionally, integrative analysis of MARCKS was conducted in the CSIOVDB dataset, which includes transcriptomic profiles of 3,431 ovarian cancer
Figure 1: The upregulation and significance of MARCKS in ovarian tumor stroma. A. Graphical representation of
computational analysis using the microdissected stromal profiling datasets of high grade EOC (GSE40595) and invasive breast cancer (GSE9014). B. Normalized expression of MARCKS in microdissected tumor stroma versus matched microdissected normal stromal tissues (15 samples in GSE22863 of lung cancer and 6 samples in GSE26910 of prostate cancer). C. Western blot analysis of MARCKS and αSMA in normal ovary stromal tissues and high grade ovarian tumor stromal tissues. SKOV3 served as a negative and MRC5 served as a positive control for stromal molecules expression. GAPDH was used as the loading control. D. Comparison of MARCKS expression in microdissected tumor stromal tissues with that of microdissected tumor epithelial tissues using three EOC profiles (GSE9890, GSE40595 and GSE38666). E. Comparison of MARCKS expression in microdissected tumor epithelial tissues with that of microdissected normal epithelial tissues using two EOC profiles (GSE40595 and GSE38666). F. Normalized expression of MARCKS in primary tumors and the metastases using two EOC profiling data (GSE2109 and GSE9891). G. Normalized expression of MARCKS in EOC tumor samples obtained before and after chemo-intervention using the GSE15622 dataset. www.impactjournals.com/oncotarget
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MARCKS was primarily restricted to stromal CAFs and it served as an independent prognostic factor of poor outcome in various cancers.
MARCKS on CAF features, we used MARCKS-specific small interfering RNA (siRNA) and the PKC inhibitor enzastaurin (Enza) to attenuate MARCKS in MRC5 fibroblast cell line-induced CAFs (MRC5-CAFs). Western blot confirmed that MARCKS and p-MARCKS proteins were significantly suppressed with MARCKS siRNA or Enza intervention (Figure 3B). Cell viability assays revealed that CAF proliferation was notably attenuated after MARCKS inhibition (Figure 3C). In addition, CAF cells were much more sensitive to cytotoxic agents such as cisplatin (cDDP) or taxol (Figure 3D). Similarly, CAF cells showed a diminished migratory capacity after silencing of MARCKS (Figure 3E). As the developmental program epithelial-mesenchymal transition (EMT) correlates with malignant cells migration and chemosensitivity [39], we analyzed MARCKS expression with EMT associated signature genes and found a strong positive relationship between them in large profiling cohorts of ovarian cancer
MARCKS facilitates proliferation, chemotherapeutical resistance and migration of CAFs In accordance with the above finding that MARCKS was elevated after chemotherapeutical intervention, we performed immunoblotting in stromal fibroblasts isolated from OC tumor tissues before and after chemo-treatment and found that MARCKS protein was drastically induced by cytotoxic agents (Figure 3A). Given the increase in MARCKS expression as OC progresses or under chemo-intervention, we hypothesized a potential role of MARCKS in OC metastasis and disease recurrence by activating tumor stroma. To explore the impact of
Figure 2: Immunohistochemical staining of MARCKS in ovarian tissues and its clinical relevance. A. Immunohistochemistry (IHC) detection of MARCKS in a series of ovarian tissues including normal ovaries, normal fallopian tubes, paired primary and metastatic tumor tissues of high grade EOC patients. B. Scoring of MARCKS in the epithelial and stromal compartment of the above IHC stained tissues. Gene expression profiles of MARCKS in ovarian cancer patients according to disease state C. FIGO stage D. clinical response E. in the CSIOVDB dataset of ovarian cancer [37]. Abbreviation: OSE, ovarian surface epithelium; FTE, fallopian tube epithelium; Mets, metastasis. Meta-analysis depicting the forest plot of MARCKS expression as a univariate predictor of overall survival (OS) F. and progression-free survival (PFS) G., using several datasets with applicable genes expression and survival information of high grade EOC patients. (**P
www.impactjournals.com/oncotarget/
Research Paper
MARCKS contributes to stromal cancer-associated fibroblast activation and facilitates ovarian cancer metastasis Zongyuan Yang1,*, Sen Xu1,*, Ping Jin1, Xin Yang1, Xiaoting Li1, Dongyi Wan1, Taoran Zhang1, Sixiang Long1, Xiao Wei1, Gang Chen1, Li Meng1, Dan Liu1, Yong Fang1, Pingbo Chen1, Ding Ma1, Qinglei Gao1 1
ancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, C Wuhan, Hubei 430030, China
*
These authors have contributed equally to this work
Correspondence to: Ding Ma, email: [email protected] Qinglei Gao, email: [email protected] Keywords: ovarian cancer, CAFs, MARCKS, senescence, Twist1 Received: January 07, 2016 Accepted: March 28, 2016 Published: April 13, 2016
ABSTRACT The Cancer Genome Atlas network has revealed that the ‘mesenchymal’ epithelial ovarian cancer (EOC) subtype represents the poorest outcome, indicating a crucial role of stromal cancer-associated fibroblasts (CAFs) in disease progression. The cooperative role of CAFs in EOC metastasis has long been recognized, but the mechanisms of stromal CAFs activation are still obscure. Therefore, we carried out an integrative analysis to identify the regulator genes that are responsible for CAFs activation in microdissected tumor stroma profiles. Here, we determined that myristoylated alanine-rich C-kinase substrate (MARCKS) was highly expressed in ovarian stroma, and was required for the differentiation and tumor promoting function of CAFs. Suppression of MARCKS resulted in the loss of CAF features, and diminished role of CAFs in supporting tumor cell growth in 3D organotypic cultures and in murine xenograft model. Mechanistically, we found that MARCKS maintained CAF activation through suppression of cellular senescence and activation of the AKT/ Twist1 signaling. Moreover, high MARCKS expression was associated with poor patient survival in EOC. Collectively, our findings identify the potential of MARCKS inhibition as a novel stroma-oriented therapy in EOC.
“proliferative,” and “mesenchymal,” in a cohort of 557 serous EOC patients [5]. A subsequent follow-up study determined that patients with the “mesenchymal” subtype presented the worst prognosis [7]. These newly emerged classification schemes based on molecular profiling facilitate our understanding of EOC heterogeneity and the development of personalized treatment strategies [8-10]. Moreover, this advantageous stratification emphasized the importance of tumor microenvironment, especially in terms of the stromal infiltrating components in EOC patients. The most prominent cell types in the tumor microenvironment are the cancer-associated fibroblasts (CAFs), which primarily contributed to the assignment of the “mesenchymal” cluster [11, 12]. CAFs are heterogeneous populations that include myofibroblasts and reprogrammed variant normal tissue derived cells such as
INTRODUCTION High-grade epithelial ovarian cancer (EOC) remains the most lethal gynecological cancer and exhibits considerable heterogeneity [1]. Traditional histopathological classification of EOC into “serous,” “mucinous,” “clear cell,” and “endometrioid” subtypes was limited in guiding therapy decisions [2, 3]. To complement conventional histopathology, molecular classification based on large-scale gene profiling was carried out and enabled the discovery of several EOC subtypes [4-6]. Tothill et al. first reported six molecular categories in 285 EOC patients and found that tumors expressing a reactive stromal gene signature were associated with a poor prognosis [4]. More recently, The Cancer Genome Atlas (TCGA) project described four subtypes, namely, “immunoreactive,” “differentiated,” www.impactjournals.com/oncotarget
37649
Oncotarget
fibroblasts and endothelial and mesothelial cells in EOC [13-15]. Generally, CAFs support cancer cells through both cell-to-cell contact interactions and soluble factors via secretion of cytokines, chemokines and ECM (Extracellular matrix) components [16-18]. The cooperative role of CAFs in EOC cell proliferation, adhesion and metastasis has long been recognized, but the mechanisms involved in stromal CAF activation are still largely unknown [18-20]. Previous studies have emphasized that perpetual activation of stromal CAFs is indispensable for tumor expansion [14-19]. Therefore, understanding the molecular profile of activated CAFs could help in targeting this major accessory of tumor microenvironment. Recent molecular investigation has identified a “stromalresponse” signature that predicts poor prognosis [21] and defined a “reactive stroma signature” characterizing primary chemoresistance in EOC [22]. Although previous studies sought to characterize specific ovarian tumor stromal genes in a compartmentalized fashion, the samples used were whole tumor specimens, which cannot exclude the interference of the epithelial compartment. Meanwhile, the recently discovered stromal signature genes are more likely to be downstream functional genes than the upstream regulator genes. These factors prompted us to explore the underlying regulators that control the active CAF signature in pure tumor stromal tissues. The emergence of specialized microdissected stroma profiling data allows the identification of tumor stroma gene signatures, as well as the potent regulator genes controlling stroma activation [18, 23-25]. To identify the regulators of stromal CAF activation, we employed the most widely accepted marker of the CAF phenotype— αSMA, which constitutes a stress fiber system bridging communication between CAFs and the ECM [26]. Thus, we carried out an integrative analysis of microdissected stromal gene profiles of EOC and invasive breast tumors [18, 25]. Among the genes identified, myristoylated alanine-rich C-kinase substrate (MARCKS) was found to be highly associated with αSMA expression in EOC tumor stroma, and was notably overexpressed in tumor stroma of both ovarian and invasive breast cancer. MARCKS, originally identified as a major target of protein kinase C (PKC), is a key regulatory molecule regulating actin dynamics [27]. Recently, it has been shown to play a fundamental role in mediating chemoresistance of breast and lung cancer [28, 29]. There are limited studies examining MARCKS in EOC metastasis. Although MARCKS has been reported to promote fibroblast migration [30], the role of MARCKS on CAF traits and the underlying mechanism involved is not well understood. This study demonstrates elevated stromal expression of MARCKS along with OC advancement, and shows that MARCKS sustains the CAF features through suppression of cellular senescence and maintenance of AKT signaling. Suppression of MARCKS attenuates CAF activity and www.impactjournals.com/oncotarget
their tumor-supporting role in 3D organotypic culture and an OC murine xenograft model. A meta-analysis of a total of 2970 serous OC expression profiles confirmed MARCKS as a prognostic factor of poor patient outcome. Our results address the role of MARCKS in the tumor stroma as a pivotal regulator of CAF activation. Thus, MARCKS could be an attractive target for stroma-oriented therapy in EOC patients.
RESULTS Overexpression and significance of MARCKS in tumor stromal fibroblasts To explore the regulatory molecules that drive gene expression representative of CAF features in EOC, we carried out an integrative analysis to identify genes that are important for CAF activation and specifically upregulated in tumor stroma. Currently, the most definitive molecular marker of CAFs is αSMA, which indicates activation of normal fibroblasts and plays a critical role in mediating communication between the stromal cells and the matrix [26, 31]. Here, we identified a cluster of 503 genes (Supplementary Table S1) that were notably positively correlated with αSMA expression in dataset GSE40595 that includes microdissected ovarian profiling data, of which fibroblasts were shown to be the major constituent [32]. By analyzing overlapping genes with the calculated 784 genes (Supplementary Table S2) in ovarian tumor stroma and 468 genes (Supplementary Table S3) in breast tumor stroma that significantly upregulated compared with their normal fibroblast counterparts in GSE40595 and breast stromal profile GSE9014, we identified ARID4B, COL3A1 and MARCKS as candidate targets for controlling stromal activation (Figure 1A). Among the genes identified, ARID4B expression was not correlated with EOC patient survival (Supplementary Figure 1A and 1B). In contrast, higher COL3A1 expression was notably correlated with worse patient outcome (Supplementary Figure 1C and 1D). However, the role of collagen family members in remodeling ECM were well studied [33], and they were more likely to be the downstream functional executors than the upstream regulators of stromal activation. MARCKS was selected for further study due to its reported role in regulating tumor cell adhesion and migration [34-36]; its impact on CAFs activity has not been well studied. The advantageous expression of MARCKS in cancer stroma was further confirmed in another two stoma profiling datasets of lung and prostate cancer and in tumor stroma of EOC patient samples (Figure 1B and 1C). Compared with its expression in the tumor epithelial compartment, MARCKS was noted specifically expressed in the stromal compartment as determined by analysis of EOC-related datasets (Figure 1D). In contrast, MARCKS level was reduced in the tumor epithelial cells compared with normal ovary epithelial 37650
Oncotarget
tissues (Figure 1E). Data mining in EOC profiling data also showed that MARCKS expression was elevated along with disease metastasis (Figure 1F) or after chemointervention (Figure 1G). All these data demonstrated that MARCKS was highly expressed in ovarian stroma and might regulate the stromal CAF activity.
specimens [37]. We found that MARCKS expression was elevated in tumor stroma and reduced in tumor epithelia as OC progresses (Figure 2C), and was increased in patients with a higher FIGO stage (Figure 2D) or in those that developed chemoresistance or disease recurrence (Figure 2E). Next, we assessed the effect of tumoral MARCKS expression on patient survival. Firstly, we analyzed its significance in a large set of EOC samples included in curatedOvarianData [38], which includes expression data as well as survival information. MARCKS mRNA expression was found to be significantly correlated with poor patient overall survival (OS) (HR=1.11, p=1.35e-4) (Figure 2F), and with worse progression-free survival (PFS) in EOC patients (HR=1.07, p=0.0499) (Figure 2G). The impact of MARCKS on patients survival was further analyzed in a large annotated database of breast and lung cancer patient samples. Kaplan–Meier analyses showed that elevated MARCKS was significantly correlated with decreased OS (HR=1.28, p=0.039), post-progression survival (PPS) (HR=1.63, p=0.00066) and relapse-free survival (RFS) (HR=1.59, p=2.1e-14) (Supplementary Figure 2A) in breast cancer and with decreased OS (HR=1.3, p=5.7e-05), PPS (HR=1.46, p=0.008) and first progression (FP) (HR=1.95, p=1.5e-10) (Supplementary Figure 2B) in lung cancer. These findings indicated that
MARCKS is specifically expressed in stromal CAFs and correlates with patient outcome To further validate the expression pattern of MARCKS during OC progression, we analyzed MARCKS expression in tissue samples including 10 normal ovarian tissues, 12 normal fallopian tube tissues and 18 pairs of primary and metastatic EOC tissues. Immunohistochemistry (IHC) demonstrated that 65% of normal ovary and 72% of the fallopian tube tissue versus 18% of the primary and 27% of the metastatic tumor samples showed moderate to strong epithelial MARCKS immunostaining, whereas 68% of the primary and 95% of the metastatic tumor sample versus 14% of the normal ovary and 32% of the fallopian tube tissues showed moderate to strong stromal MARCKS immunostaining. (Figure 2A and 2B). Additionally, integrative analysis of MARCKS was conducted in the CSIOVDB dataset, which includes transcriptomic profiles of 3,431 ovarian cancer
Figure 1: The upregulation and significance of MARCKS in ovarian tumor stroma. A. Graphical representation of
computational analysis using the microdissected stromal profiling datasets of high grade EOC (GSE40595) and invasive breast cancer (GSE9014). B. Normalized expression of MARCKS in microdissected tumor stroma versus matched microdissected normal stromal tissues (15 samples in GSE22863 of lung cancer and 6 samples in GSE26910 of prostate cancer). C. Western blot analysis of MARCKS and αSMA in normal ovary stromal tissues and high grade ovarian tumor stromal tissues. SKOV3 served as a negative and MRC5 served as a positive control for stromal molecules expression. GAPDH was used as the loading control. D. Comparison of MARCKS expression in microdissected tumor stromal tissues with that of microdissected tumor epithelial tissues using three EOC profiles (GSE9890, GSE40595 and GSE38666). E. Comparison of MARCKS expression in microdissected tumor epithelial tissues with that of microdissected normal epithelial tissues using two EOC profiles (GSE40595 and GSE38666). F. Normalized expression of MARCKS in primary tumors and the metastases using two EOC profiling data (GSE2109 and GSE9891). G. Normalized expression of MARCKS in EOC tumor samples obtained before and after chemo-intervention using the GSE15622 dataset. www.impactjournals.com/oncotarget
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MARCKS was primarily restricted to stromal CAFs and it served as an independent prognostic factor of poor outcome in various cancers.
MARCKS on CAF features, we used MARCKS-specific small interfering RNA (siRNA) and the PKC inhibitor enzastaurin (Enza) to attenuate MARCKS in MRC5 fibroblast cell line-induced CAFs (MRC5-CAFs). Western blot confirmed that MARCKS and p-MARCKS proteins were significantly suppressed with MARCKS siRNA or Enza intervention (Figure 3B). Cell viability assays revealed that CAF proliferation was notably attenuated after MARCKS inhibition (Figure 3C). In addition, CAF cells were much more sensitive to cytotoxic agents such as cisplatin (cDDP) or taxol (Figure 3D). Similarly, CAF cells showed a diminished migratory capacity after silencing of MARCKS (Figure 3E). As the developmental program epithelial-mesenchymal transition (EMT) correlates with malignant cells migration and chemosensitivity [39], we analyzed MARCKS expression with EMT associated signature genes and found a strong positive relationship between them in large profiling cohorts of ovarian cancer
MARCKS facilitates proliferation, chemotherapeutical resistance and migration of CAFs In accordance with the above finding that MARCKS was elevated after chemotherapeutical intervention, we performed immunoblotting in stromal fibroblasts isolated from OC tumor tissues before and after chemo-treatment and found that MARCKS protein was drastically induced by cytotoxic agents (Figure 3A). Given the increase in MARCKS expression as OC progresses or under chemo-intervention, we hypothesized a potential role of MARCKS in OC metastasis and disease recurrence by activating tumor stroma. To explore the impact of
Figure 2: Immunohistochemical staining of MARCKS in ovarian tissues and its clinical relevance. A. Immunohistochemistry (IHC) detection of MARCKS in a series of ovarian tissues including normal ovaries, normal fallopian tubes, paired primary and metastatic tumor tissues of high grade EOC patients. B. Scoring of MARCKS in the epithelial and stromal compartment of the above IHC stained tissues. Gene expression profiles of MARCKS in ovarian cancer patients according to disease state C. FIGO stage D. clinical response E. in the CSIOVDB dataset of ovarian cancer [37]. Abbreviation: OSE, ovarian surface epithelium; FTE, fallopian tube epithelium; Mets, metastasis. Meta-analysis depicting the forest plot of MARCKS expression as a univariate predictor of overall survival (OS) F. and progression-free survival (PFS) G., using several datasets with applicable genes expression and survival information of high grade EOC patients. (**P
